Lipid homeostasis is essential for cell function and disruptions to lipid homeostasis lead to disease. Elevated serum cholesterol is a primary risk factor for heart disease and atherosclerosis, a leading killer of adults in the United States. Fatt acid and triglyceride accumulation in the liver causes fatty liver that frequently progresses to non-alcoholic steatohepatitis, liver cirrhosis and cancer. Type II diabetes mellitus is a major ris factor for developing fatty liver as excess serum glucose is converted into fatty acid by the liver Alarmingly, diabetes is projected to affect one-quarter of the U.S. population by 2050. Knowing how cellular lipid homeostasis is regulated will identify therapeutic opportunities for these increasingly common diseases. Membrane-bound, basic helix-loop-helix leucine zipper transcription factors called sterol regulatory element-binding proteins (SREBPs) are the central regulators of cellular lipid homeostasis, controlling synthesis and uptake of cholesterol, fatty acids, and triglycerides. Using fission yeast, we discovered that the SREBP pathway is conserved in fungi, controlling adaptation to low oxygen in addition to lipid homeostasis. Fission yeast SREBP is proteolytically activated through a novel pathway that requires the Golgi Dsc E3 ligase and the AAA-ATPase Cdc48. Our studies in Cryptococcus neoformans and in Aspergillus fumigatus by others demonstrated that this oxygen-responsive pathway is conserved across fungal phyla, and that the SREBP pathway is essential for virulence in these important opportunistic human fungal pathogens. Thus, our studies impact research of lipid homeostasis, protein and degradation, and fungal pathogenesis. In this proposal, we will continue our studies of the SREBP pathway and the Dsc E3 ligase to understand how cells regulate lipid homeostasis in response to changes in environmental oxygen. We hypothesize that the Dsc E3 ligase ubiquitinates SREBP in the Golgi to target it to Rbd2-Cdc48 for cleavage and membrane release. To test this hypothesis, we propose the following specific aims: AIM 1. TO IDENTIFY REQUIREMENTS FOR SREBP BINDING TO DSC E3 LIGASE. AIM 2. TO TEST WHETHER CLEAVAGE REQUIRES SREBP UBIQUITINATION. AIM 3. TO TEST WHETHER RBD2 IS A SREBP PROTEASE. AIM 4. TO DETERMINE THE FUNCTION OF CDC48 IN SREBP CLEAVAGE. The long-term goal of this project is to use fission yeast as a discovery tool to identify new mechanisms for regulation of SREBPs in mammalian cells and new targets for SREBP pathway inhibition toward treatments of fungal disease. To date, our work has highlighted environmental oxygen as a key regulator of lipid synthesis. The expectation is that our proposed studies will describe new mechanisms for how SREBPs are recognized by E3 ligases for ubiquitination and by rhomboid proteases for cleavage advancing our understanding of the SREBP pathway and diseases of lipid homeostasis.